The present invention relates generally to hematology and the molecular biology of blood growth factors, and specifically to the use of interleukin-7 in medicine as an inducer of megakaryocyte differentiation.
Megakaryocytes are the cellular source of platelets and arise from a common bone marrow precursor cell which gives rise to all hematopoietic cell lineages. The common precursor cell, known as the pluripotential hematopoietic stem cell, or PHSC, gives rise to the burst forming unit megakaryocyte (BFU-MK) which responds to IL-3 and tumor promoting phorbol esters (e.g., phorbol myristate acetate, PMA) in vitro, inducing development of large multi-focal colonies of megakaryocytes (MK). BFU-MK differentiate into MK colony forming cells (CFU-MK) which form colonies in vitro in response to IL-3, GM-CSF, EPO, G-CSF, IL-6 or IL-4, although evidence for the latter four is controversial. CFU-MK give rise to morphologically recognizable small MK which in the mouse stain with acetylcholinesterase (SAChE+ cells). It is unclear whether these cells have any proliferative potential remaining. SAChE+ cells then undergo a series of cytoplasmic and nuclear maturation steps (polyploidization) ultimately resulting in platelet producing MK. In vivo evidence suggests that the proliferative steps at the level of MK colony forming cells and the maturation steps resulting in platelet production are independently regulated by distinct cytokines.
Urine, serum and plsma from patients with hypomegakaryocytic thrombocytopenia have been shown to promote the formation of CFU-MK by bone marrow mononuclear cells in vitro. See, e.g., Kawakita et al., Br. J. Haematol. 52:429; Hoffman et al., N. Engl. J. Med. 305:533 (1981); Messner at al., J. Cell. Physiol. Supp. 1:45 (1982); and Kimura et al., J. Cell. Physiol. 118:87-96. None of these putative factors were purified to a significant degree.
Hoffman et al., J. Clin. Invest. 75:1174 (1985) reported purification of a megakaryocyte colony stimulating factor, designated Meg-CSF, from the sera of patients with hypomegakaryocytic thrombocytopenia. Ammonium sulfate precipitation, DEAE-cellulose chromatography, lectin affinity chromatography and RP-HPLC provided a glycoproteinaceous material which exhibited a molecular weight of about 46,000 daltons (da) and which stimulated the development of CFU-MK in bone marrow assays. This material has never been cloned or sequenced, and only modest effects in vivo have ever been reported.
Rosenberg, European Patent Application 260,918, discloses a putative "megakaryocyte stimulating factor" elaborated by human embryonic kidney cells which is alleged to be a specific hematopoietin for cells of the megakaryocyte lineage. The purified fraction disclosed in this reference was an "acidic protein" having an apparent molecular weight of about 15,000 in an assay in which protein synthesis by a partially purified megakaryocyte fraction and a rat promegakaryoblast cell line was determined. No in vivo studies were reported, and the extant literature has not reported further progress in cloning or sequencing this factor.
Interleukin-7 (IL-7), also known as lymphopoetin-1, is a lymphopoietic growth factor first isolated and cloned by virtue of its ability to stimulate growth of B and T cell progenitors in bone marrow. PCT Application U.S. Ser. No. 88/03747, filed Oct. 19, 1988, and European Patent Application No. 88309977.2, filed Oct. 24, 1988, (see also U.S. Ser. No. 07/113,566 now abandoned) disclose DNAs, vectors, and related processes for producing mammalian IL-7 proteins by recombinant DNA technology. The relevant disclosures of these patent applications are hereby incorporated by reference. The cloning of murine IL-7 was first reported by Namen et al., Nature 333:571 (1988) and human IL-7 by Goodwin et al., Proc. Natl. Acad. Sci. USA (in press). Purification of murine IL-7 from supernatants of a transformed bone marrow stromal cell line indicated an apparent molecular weight of approximately 25,000 da. See Namen et al., J. Exp. Med. 167:988 (1988). The cloned DNAs reported by Namen and Goodwin suggest minimum molecular weights for the murine and human molecules of 14,897 and 17,387 daltons, respectively, exclusive of any glycosylation.
It has now been found that IL-7 has the ability to significantly stimulate platelet production (thrombocytopoiesis) in vivo. This property of the molecule should render it a useful adjunct in therapy of patients suffering from acute thrombocytopenia, for example, as a result of chemo- or radiotherapy of various cancers. Currently, such patients are at grave risk when circulating platelet levels are depressed to levels wherein thrombogenesis is precluded. Conventional therapy for acute life-threatening thrombocytopenia involves repeated transfusions of purified platelets.